KR20200011927A - Compounds for treating diseases associated with mitochondrial dysfunction - Google Patents

Abstract

.The present invention relates to a compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, for use in the prevention or treatment of a disease associated with mitochondrial dysfunction:

.

Description

Compounds for treating diseases associated with mitochondrial dysfunction

The present invention relates to compounds, pharmaceutical compositions and dietary supplements useful for the prevention or treatment of diseases associated with mitochondrial dysfunction, especially Parkinson's disease.

Parkinson's disease (PD), the most common motor disorder and the second most common neurodegenerative disease after Alzheimer's disease, is characterized mainly by the loss of dopamine-functional neurons in the dentin, causing dopamine defects in the striatum. The resulting dysregulation of the basal ganglia circuit is the reason for the most important motor symptoms, including laxity, hypothyroidism, stiffness, stabilization and postural instability. In addition to typical motor symptoms, various non-motor characteristics can be developed, such as autonomic ataxia, sleep disorders, depression and cognitive impairment, which show a broader degeneration process.

Little is known about the causative pathology of PD. The most common sporadic form of PD appears to be a complex multifactorial disorder with variable contributions from environmental factors and genetic susceptibility, with aging being the most important risk factor.

Current treatment of PD aims to correct the consequences of dopaminergic neuron loss by increasing dopamine concentrations via L-DOPA administration. L-DOPA crosses the protective blood-brain-barrier, while dopamine itself cannot, and once L-DOPA enters the central nervous system, it is converted to dopamine by the enzyme DOPA decarboxylase. Pyridoxal phosphate (vitamin B6) is a necessary cofactor of this reaction and can sometimes be administered in the form of pyridoxine, usually with L-DOPA.

However, in the treatment of Parkinson's disease, chronic L-DOPA administration may be associated with end-of-dose deterioration of function, on / off oscillation, and freezing during movement. Induces several unwanted side effects such as dyskinesia at peak dose and dopamine dysregulation syndrome, as well as drug resistance (Thanvi & Lo (2004) Postgrad. Med. J. 80 : 452-458) .

Thus, there is a need for alternative treatment for L-DOPA administration in Parkinson's disease.

Summary of the Invention

Recent studies on the function and dysfunction of PD-associated genes have provided fundamental new insights into the biochemical pathways associated with disease processes. This finding establishes that mitochondrial dysfunction is a common denominator of sporadic and familial PD, moving mitochondria to the forefront of PD research (Winklhofer & Haass (2010) Biochimica et Biophysica Acta 1802 : 29-44).

By the present inventors, the present invention arises from the unexpected finding that queuine has a neuroprotective effect in an in vitro model of Parkinson's disease, including mitochondrial dysfunction.

Accordingly, the present invention provides a quiein, a precursor of quieuin, a derivative of quieuin, an analog of quieuin, or a stereoisomer of quieuin for use in the prevention or treatment of a disease associated with mitochondrial dysfunction, or To pharmaceutically acceptable salts or hydrates thereof.

The invention also relates to a compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, for use in the prevention or treatment of a disease associated with mitochondrial dysfunction in an individual:

[In meals:

R ₁ represents -H or a ribosil group of the formula:

{In the expression:

R ₆ is -H; -OR ₉ or -O-CO-R ₉ , wherein R ₉ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms;

R ₇ is -H; -OR ₁₀ or -O-CO-R ₁₀ (R ₁₀ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms); Deoxyribonucleic acid groups; Or a ribonucleic acid group;

R ₈ is -H; -OR ₁₁ or -O-CO-R ₁₁ (R ₁₁ is H, an alkyl group having 1 to 20 carbon atoms or an aryl group having 3 to 20 carbon atoms); Phosphate groups; Diphosphate groups; Triphosphate groups; Deoxyribonucleic acid groups; Or a ribonucleic acid group};

R ₁₂ represents a saturated or unsaturated alkyl, cycloalkyl, heterocycloalkyl or ether group having 1 to 20 carbon atoms, optionally substituted by at least one group selected from the group consisting of:

Alkyl groups having 1 to 20 carbon atoms,

Aryl or heteroaryl groups having 3 to 20 carbon atoms,

A cycloalkyl or heterocycloalkyl group having 3 to 20 carbon atoms,

Hydroxyl group,

Carbonyl or carboxyl groups having 1 to 20 carbon atoms,

ㆍ epoxy group,

A -OR ₄ group (R ₄ is H, an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group),

-O-CO-R ₅ group (R ₅ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms or a glycosyl group).

The invention also relates to a compound of formula (A) of formula (I), or a pharmaceutically acceptable salt or hydrate thereof, for use in the prevention or treatment of a disease associated with mitochondrial dysfunction:

[In meals:

- a represents a double bond or an epoxy group,

R ₁ represents -H or a ribosil group of the formula:

{In the expression:

R ₆ is -H; -OR ₉ or -O-CO-R ₉ , wherein R ₉ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms;

R ₇ is -H; -OR ₁₀ or -O-CO-R ₁₀ (R ₁₀ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms); Deoxyribonucleic acid groups; Or a ribonucleic acid group;

R ₈ is -H; -OR ₁₁ or -O-CO-R ₁₁ (R ₁₁ is H, an alkyl group having 1 to 20 carbon atoms or an aryl group having 3 to 20 carbon atoms); Phosphate groups; Diphosphate groups; Triphosphate groups; Deoxyribonucleic acid groups; Or a ribonucleic acid group};

The same or different R ₂ and R ₃ are —OR ₄ (R ₄ is H, an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group); Or —O—CO—R ₅ (R ₅ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms or a glycosyl group).

In one embodiment of the invention, a compound of formula (A), in particular a compound of formula (I), or a pharmaceutically acceptable salt or hydrate thereof, for use as defined above, can be used for the prevention of diseases associated with mitochondrial dysfunction or In combination with at least one additional compound useful for treatment.

The invention also provides a compound of formula (A) as defined above, in particular with formula (I), optionally in combination with at least one pharmaceutically acceptable excipient or vehicle, for use in the prevention or treatment of diseases associated with mitochondrial dysfunction. A pharmaceutical composition comprising, as an active substance, a compound of the present invention, or a pharmaceutically acceptable salt or hydrate thereof.

In one embodiment of the invention, the pharmaceutical composition as defined above further comprises at least one additional compound useful for the prevention or treatment of diseases associated with mitochondrial dysfunction.

The invention further comprises at least one additional compound useful for the prophylaxis or treatment of diseases associated with mitochondrial dysfunction, optionally in combination with at least one pharmaceutically acceptable excipient or vehicle. A pharmaceutical composition comprising as an active substance a compound of A), in particular a compound of formula (I), or a pharmaceutically acceptable salt or hydrate thereof.

The invention also relates to a product comprising the following as a combined agent for simultaneous, separate or sequential use in the prevention or treatment of a disease associated with mitochondrial dysfunction in an individual:

-A compound of formula (A) as defined above, in particular a compound of formula (I), or a pharmaceutically acceptable salt or hydrate thereof,

At least one additional compound useful for the prevention or treatment of a disease associated with mitochondrial dysfunction.

The invention also includes a compound of formula (A) as defined above, in particular a compound of formula (I), or a pharmaceutically acceptable salt or hydrate thereof, for use in reducing the risk of a disease associated with mitochondrial dysfunction. It is about a health supplement.

In one embodiment of the invention, the dietary supplement as defined above optionally comprises an additional compound selected from the group consisting of vitamins, minerals, fatty acids, amino acids and antioxidants.

The present invention also includes administering to an individual an effective amount of a compound of formula (A), in particular a compound of formula (I), or a pharmaceutically acceptable salt or hydrate thereof, as defined above. A method for preventing or treating a disease associated with dysfunction.

In one embodiment of the invention, the method as defined above further comprises administration of at least one compound useful for the prevention or treatment of a disease associated with mitochondrial dysfunction.

The invention also relates to the use of a compound of formula (A) as defined above, in particular a compound of formula (I), for the manufacture of a medicament intended for the prevention or treatment of a disease associated with mitochondrial dysfunction in an individual. will be.

In one embodiment of the invention, the medicament as defined above further comprises at least one compound useful for the prevention or treatment of diseases associated with mitochondrial dysfunction.

Detailed description of the invention

Compound of Formula (A)

Compounds of formula (A) as defined above, in particular compounds of formula (I), are in particular described in Barnett & Grubb (2000), Tetrahedron 56: 9221-9225, Oxenford et al . (2004) Tetrahedron Lett ers 45 : 9053-9055, Brooks et al . (2010) Tetrahedron Letters 51: 4163-4165, Gerber et al. (2012) Org. Biomol. Chem. 10 : 8660-8668, Thesis by Allen Brook, titled " Synthesis of Tritium Labeled Queuine, PreQ ₁ and Related Azide Probes Toward Examining the Prevalence of Queuine" (2012, University of Michigan), Akimoto et al . (1986) J. Med. Chem. , 29 : 1749-1753, Kelly et al. (2016) Nucleic Acids Research , 1-11, and international application WO2016 / 050806, all of which are incorporated herein by reference, can be readily synthesized chemically by one of ordinary skill in the art.

Briefly, for example, Quiouin can be synthesized according to the following reaction scheme:

.

.

In addition, the compounds of formula (A) as defined above, in particular compounds of formula (I), may be prepared from natural sources such as microorganisms, in particular bacteria, or from plants, in particular alpha-Proteobacteria such as Rhizobium. , Mesorhizobium and Sinorhizobium genii can be extracted and optionally purified from plants with nodes to bacteria.

For example, queosine can be obtained from tRNAs prepared as follows, in particular tRNA ^Asn , tRNA ^Asp , tRNA ^His and tRNA ^Tyr :

Preparation of Total RNA Under Acidic Conditions

B. subtilis strains (or other related bacteria) are grown in ED liquid medium with the appropriate supplement at 37 ° C. with constant aeration. Fresh overnight cultures are inoculated with 15 ml of ED medium at an optical density of 0.1 at 600 nm (OD600). Cells are grown at 37 ° C. with an OD600 of 1 and cooled at −80 ° C. in 60% methanol in equal volume of 70 mM Hepes, pH 7.5. All subsequent steps are run under cold and the solution for the crude RNA prepared is treated with diethyl pyrocarbonate and sterilized. Cells are pelleted at 4 ° C., washed in water and resuspended in 0.5 ml of 10% glucose, 11 mM Tris, 10 mM EDTA. The suspension is transferred to a tube containing 0.1 g glass beads (acid-washed) (sigma-Aldrich, G4649). The tube is placed in The CoolPrep Adapter of FastPrep® 24 Instrument (MP Biomedicals) containing 50 g of dry ice. Cells are destroyed after 3 cycles using the following parameters: 6 m / s for 45 seconds. After each cycle, the suspension is kept on ice for 1 minute. After 2 min centrifugation at 10,000 rpm, the supernatant is transferred to a new Eppendorf tube. 0.3 M sodium acetate pH 5.2 is added and total RNA is isolated under acidic conditions. One volume of acid phenol: chloroform and isoamyl alcohol (125: 24: 1) pH 4.5 (Amresco, AM9720) are added. The samples are mixed by vortexing for 10 seconds and incubated in a 65 ° C. water bath for 3 minutes. The phases are separated by spinning at 14,000 rpm for 5 minutes, then the aqueous phase is reextracted once with the same hot acid phenol procedure. Transfer the aqueous phase to a new tube and replenish with 1 volume of cooled acid phenol. After centrifugation at 14,000 rpm for 5 minutes, RNA is precipitated with 2.5 volumes of anhydrous ethanol at -80 ° C for 1 hour. RNA is pelleted at 4 ° C. at 14,000 rpm for 15 minutes and washed with 70% ethanol. RNA pellets are dissolved in 10 mM Tris, 1 mM EDTA pH 7.5.

enrichment of tRNA

The total RNA preparation is mixed with 1 volume of lithium chloride 8 M pH 4.5 and sodium acetate pH 5.2 at 0.01 mM final concentration. This RNA solution is incubated at -80 ° C for 2 hours. After centrifugation at 14,000 rpm for 15 minutes at 4 ° C, tRNA remained in the supernatant. To remove salt contamination, tRNA is precipitated at −80 ° C. for 1 hour by adding 0.3 M sodium acetate pH 5.2 and 2.5 volumes of anhydrous ethanol. The tRNA is then pelleted by centrifugation at 14,000 rpm for 15 minutes at 4 ° C. and washed with 70% ethanol. tRNA pellets are dissolved in 10 mM Tris, 1 mM EDTA pH 7.5.

The stereoisomers of the quiein in accordance with the present invention may be of any type. Preferably, the stereoisomer of the quiein is ent-quiuein.

The pharmaceutically acceptable salts or hydrates according to the invention may be of any type. However, it is preferred that the pharmaceutically acceptable salt according to the invention is a hydrochloride salt.

Preferably, the glycosyl group according to the present invention is selected from the group consisting of mannosyl groups, galactosyl groups or glutamyl groups.

Preferably, the aminoacyl groups are alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), Glutamic Acid (glu, E), Glycine (gly, G), Histidine (his, H), Isoleucine (ile, I), Leucine (leu, L), Lysine (lys, K), Methionine (met, M), Phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V) .

Preferably, the substituents of formula (A), in particular of formula (I) according to the invention, may be linked together.

In a preferred embodiment of the compound of formula (A) as defined above:

R ₁ is H,

R ₁₂ represents a saturated or unsaturated alkyl, cycloalkyl, heterocycloalkyl or ether group having 1 to 20 carbon atoms, optionally substituted by at least one group selected from the group consisting of:

Alkyl groups having 1 to 20 carbon atoms,

Aryl or heteroaryl groups having 3 to 20 carbon atoms,

A cycloalkyl or heterocycloalkyl group having 3 to 20 carbon atoms,

Hydroxyl group,

Carbonyl or carboxyl groups having 1 to 20 carbon atoms,

ㆍ epoxy group,

A -OR ₄ group (R ₄ is H, an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group),

-O-CO-R ₅ group (R ₅ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms or a glycosyl group).

In another preferred embodiment of the compound of formula (A) as defined above:

R ₁₂ represents a group of the formula:

[In the meal,

A represents a double bond or an epoxy group,

The same or different R ₂ and R ₃ are —OR ₄ (R ₄ is H, an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group); Or —O—CO—R ₅ (R ₅ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms or a glycosyl group).

In another preferred embodiment of the compound of formula (A) as defined above:

R ₁ is H,

R ₁₂ represents a saturated or unsaturated alkyl group having 1 to 20 carbon atoms, optionally substituted by at least one hydroxyl group.

In another preferred embodiment of the compound of formula (A) as defined above, in particular the compound of formula (I):

The same or different R ₂ and R ₃ represent —OH, —O-mannosyl group, —O-galactosyl group or —O-glutamyl group;

R ₆ represents -OH;

The same or different R ₇ and R ₈ represent —OH or ribonucleic acid groups.

Preferably, when both R ₇ and R ₈ represent a ribonucleic acid group, the compound of formula (A) according to the invention, in particular the compound of formula (I), is a ribonucleoside of transfer RNA (tRNA) As included in tRNA. More preferably, the compounds of formula (A) according to the invention, in particular the compounds of formula (I), are ribonucleosides of the anticodons of tRNA, most preferably the first nucleosides of the anticodons, ie 5 'of the anticodone. Nucleoside at the wobble position of the nucleoside or anticodone. Preferred tRNAs according to the invention are selected from the list consisting of tRNA ^Asn , tRNA ^Asp , tRNA ^His and tRNA ^Tyr .

Preferably, compounds of formula (A) as defined above, in particular compounds of formula (I), are represented by the following formulas (II), (III) or (IV):

.

.

Preferably, when the compound of formula (A) according to the invention, in particular the compound of formulas (I) to (III), is included in tRNA ^Asp , R ₃ is OH and R ₂ is O-mannose.

Also preferably, when the compounds of the formula (A) according to the invention, in particular the compounds of the formulas (I) to (III), are included in the tRNA ^Tyr , R ₃ is OH and R ₂ is O-galactose.

Preferably, the compounds of formula (A) according to the invention, in particular the compounds of formula (I), are represented by the following formula (V):

.

.

As will be apparent to those skilled in the art, all stereochemical configurations of the compounds according to the invention are intended to be covered by the formulas presented herein. In particular, as intended herein, when the stereoconfiguration of the bond is not specified, the bond may represent an uplink, a downlink, and a mixture of the two, in particular any of a 1/1 mixture.

Accordingly, the compounds of the formula (A) according to the invention, in particular the compounds of the formula (I), are also enantiomers represented by optically active forms of the compounds of the formula (V), such as the following formulas (Va) and (Vb), Or mixtures thereof, in particular racemic mixtures thereof:

.

.

The compound of formula (Va) is Quiouin. Quieuin is also known as 7- (3,4-trans-4,5- cis -dihydroxy-1-cyclopenten-3-ylaminomethyl) -7-deazaguanine.

The compound of formula (Vb) is ent-quiuein.

Preferably, the compounds of the formula (A) according to the invention, in particular the compounds of the formula (I), are formulas (VI), (VIa), (VIb), (VII), (VIIa), (VIIb), ( VIII), (VIIIa), (VIIIb), (IX), (X), (Xa) or (Xb):

.

.

Also preferably, the compound of formula (A) according to the present invention is formula (XI), (XIa), (XIb), (XII), (XIII), (XIV), (XV), (XVI) or Represented by (XVII):

.

.

The compound of formula (VIa) is an epoxy also known as 7- (5- [3,4-epoxy-2,5-dihydroxycyclopent-1-yl) amino] methyl) -7-deazaguanine Quieuin.

The compound of formula (VIIa) is 2-amino-5-({[(1S, 4S, 5R) -4,5-dihydroxycyclopent-2-en-1-yl] amino} methyl) -7- ( keosine, also known as β-D-ribofuranosyl) -1,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

The compound of formula (VIIIa) is also known as 7- (5-[(3,4-epoxy-2,5-dihydroxycyclopent-1-yl) amino] methyl) -7-deazaguanosine Epoxykeosin.

Compound of formula (XI) is N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d] pyrimidin-5-yl) methyl) -2,3 -Dihydroxypropan-1-amine.

The compound of formula (XII) is N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d] pyrimidin-5-yl) methyl) -3-phenyl Propan-1-amine.

Compound (XIII) is N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d] pyrimidin-5-yl) methyl) -propan-1-amine to be.

Compound (XVI) is N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d] pyrimidin-5-yl) methyl) -butan-1-amine to be.

Compound (XVII) is N-((2-amino-4-oxo-4,7-dihydro-3H-pyrrolo [2,3-d] pyrimidin-5-yl) methyl) -hexane-1-amine to be.

Preferably, the compounds of formula (A) according to the invention, in particular the compounds of formula (I), are mannosyl-quiues, galactosyl-quiues, glutamyl-quiues, galactosyl-ke Auo, mannosyl-keosin, glutamyl-keosine, quieu-in-tRNA and epoxyquiouin-tRNA.

Also preferably, the compounds of formula (A) according to the invention, in particular the compounds of formula (I), are Quiouin-tRNA ^Asp , Quiouin-tRNA ^Tyr , Epoxyquiouin-tRNA ^Asp , Epoxyquiouin -tRNA ^Tyr , Quiuein-tRNA ^Asn , Quiuein-tRNA ^His , Epoxyquiuein-tRNA ^Asn , Epoxyquiuein-tRNA ^His , Mannosyl-Quieuein-tRNA ^Asp , Galactosyl-Quiee Iso-tRNA ^Tyr , mannosyl-epoxyquieuin-tRNA ^Asp and galactosyl-epoxyquieuin-tRNA ^Tyr .

Also most preferably, the compounds of the formula (A) according to the invention, in particular the compounds of the formula (I), are quieuin, ent-quiouin, keosin, epoxyquiouin, epoxykeosin, mannosyl- Quiuein, galactosyl-quiuein, glutamyl-quiuein, galactosyl-keosin, mannosyl-keosin, glutamyl-keosin, quieuin-tRNA, epoxyquiuein-tRNA , A compound of the formulas (XI), (XIa) and (XIb).

Diseases Associated with Mitochondrial Dysfunction

As intended herein, the expression “disease associated with mitochondrial dysfunction” includes a disease associated with or caused by a mitochondrial dysfunction, disease or disorder.

Preferably, the disease associated with mitochondrial dysfunction according to the present invention is a neuronal disease associated with mitochondrial dysfunction, more preferably a central nervous system disease associated with mitochondrial dysfunction, most preferably a neurodegenerative disease associated with mitochondrial dysfunction.

Preferably, the disease associated with mitochondrial dysfunction according to the invention is Parkinson's disease.

Parkinson's disease is well known to those skilled in the art and is specifically defined in sections G20, G21 and G22 of ICD-10 Version: 2016.

As intended herein, Parkinson's disease according to the present invention particularly includes:

Primary (idiopathic) Parkinsonism,

-Secondary Parkinsonism (acquired),

Atypical Parkinsonism, and

Familial neurodegenerative disease causing Parkinsonism.

Parkinson's disease according to the invention also includes sporadic and familial Parkinson's disease.

Parkinson's disease according to the invention in particular also includes stage I, stage II, stage III, stage IV and stage V Parkinson's disease.

individual

As intended herein, the individual according to the invention is preferably a human.

Preferably, an individual according to the invention is over 30, 40, 50, 60, 70 or 80 years old.

Also preferably, the individual according to the invention is under 80, 70, 60, 50 or 40 years old.

Also preferably, the individual according to the invention is an individual with stage I, II, III, IV or V Parkinson's disease.

Also preferably, the individual according to the present invention does not develop Parkinson's disease or show symptoms of Parkinson's disease and is at risk of developing Parkinson's disease. More preferably, the individual according to the invention does not have Parkinson's disease or does not have Parkinson's disease symptoms and at least one of his or her relatives suffers from Parkinson's disease.

Additional compounds

Additional compounds useful for the prevention or treatment of diseases associated with mitochondrial dysfunction can be of any type known to those skilled in the art. Preferably, the further compounds according to the invention are selected from the group consisting of vitamins, minerals, fatty acids, amino acids, antioxidants and derivatives or precursors thereof.

Preferably the vitamin is selected from the group consisting of pyridoxine, pyridoxal phosphate (vitamin B ₆ ), riboflavin, thiamine, vitamin E, vitamin K3, vitamin C, niacin, CoQ10 and β-carotene.

Preferably, the mineral is selected from the group consisting of calcium, magnesium, selenium and phosphorus.

Preferably, the amino acid is L-DOPA (levodopa).

Preferably, the fatty acid is selected from the group consisting of Levo-carnitine and acetyl-L-carnitine.

administration

As intended herein, “combined” or “in combination” means that a compound of formula (A) as defined above, in particular a compound of formula (I), is simultaneously, ie identically administered with another compound or product On site, or separately, or at different times, provided that the compound of formula (A) as defined above, in particular the compound of formula (I), and the time period during which its effect is exerted in the individual The time period during which the agent or product exerts its pharmacological effect in the individual crosses at least partially.

 Preferably, the compound of formula (A) according to the invention, in particular the compound of formula (I), or a pharmaceutically acceptable salt or hydrate thereof, is 0.01 to 40 mg / kg / d, more preferably 0.01 to 10 mg / kg, even more preferably 0.01 to 1 mg / kg / d, most preferably 0.01 to 0.1 mg / kg / d in a dosage regimen or for administration.

Preferably, the compounds of formula (A) according to the invention, in particular the compounds of formula (I), or pharmaceutically acceptable salts or hydrates thereof, are oral route, intradermal route, intravenous route, intramuscular route or subcutaneous route Or in a form suitable for administration. Preferably, the compound of formula (A) according to the invention, in particular the compound of formula (I), or a pharmaceutical composition, medicament, product or dietary supplement comprising the same, is administered or administered by a hypodermic implant Suitable form for

Preferably, the compounds of formula (A) according to the invention, in particular the compounds of formula (I), or pharmaceutical compositions, medicaments, products or dietary supplements comprising the same, are powders, sachets, tablets, gelatins, capsules. , Or in the form of a liquid or gel solution.

Also preferably, the pharmaceutical composition, medicament, product or dietary supplement according to the invention is a compound of formula (A) according to the invention, in particular a compound of formula (I), in particular quieuin, ent-quiouin , Keosine, or a compound of formula (XI), (XIa) or (XIb) in a unit dose of at least 0.15 mg, 1 mg, 10 mg, 50 mg, 100 mg, 500 mg or 1000 mg.

Also preferably, the pharmaceutical composition, medicament, product or dietary supplement according to the invention is a compound of formula (A) according to the invention, in particular a compound of formula (I), in particular quieuin, ent-quiouin , Microorganisms comprising keosine or a compound of formula (XI), (XIa) or (XIb) in particular in unit doses of at least 0.15 mg, 1 mg, 10 mg, 50 mg, 100 mg, 500 mg or 1000 mg And / or extracts from plants, in particular purified extracts.

1
1 is a control (grey bar, control) in the absence of 6-OHDA, and 6-OHDA alone (black bar), Quiuein (dot bar) added 1 day before 6-OHDA + 6-OHDA intoxication And survival of primary dopaminergic neuron cultures, expressed in%, of 6-OHDA + BDNF (horizontal bars). * Indicates p <0.05 for 6-OHDA.
2
FIG. 2 is a control (grey bar, control) in the absence of 6-OHDA, 6-OHDA alone (black bar), Quiuein (small dot bar) added 6 days before 6-OHDA + 6-OHDA intoxication and 6 Survival of primary dopaminergic neuron cultures, expressed in%, of -OHDA + BDNF (large dot bar). * Indicates p <0.05 for 6-OHDA.

Example

6-hydroxydopamine (6-OHDA) is not only used as a pharmacological agent capable of triggering Parkinson's disease-like stigmata, but also (Sauer and Ortel (1994) Neuroscience 59 : 401-15; Cass et al . Brain Res . 938 : 29-37), which are likely to correspond to naturally occurring dopamine-functional degradation metabolites that accumulate in Parkinson's-affected brain and appear to contribute strongly to these pathological aspects (Ellenberg et al . 1995) in " Etiology of Parkinson's Desease" , eds Ellenberg, Koller, Langston (Marcel Dekker, New York), pp 153-201; Jellinger et al . (1995) J. Neural. Transm. 46 : 297-314) Selective Catecholamines Sex neurotoxin.

For this reason, 6-OHDA-induced dopaminergic neurotoxicity in rats is widely used as a model for PD studies (Simola et al . (2007) Neurotox. Res . 11 : 151-167; Mercanti (2012) Methods Mol. Biol . 846 : 355-364; De Jesus-Cortes et al. (2015) npj Parkinson's Desease 1 : 15010. In addition, 6-OHDA has been shown to impair mitochondrial function in this rat model of Parkinson's disease, allowing detection of apparent mitochondrial dysfunction as a suitable surrogate marker for preclinical evaluation of potential neuroprotective strategies (Kupsch). et al. (2014) J. Neural.Transm. 121 : 1245-1257).

In this regard, with respect to the in vitro dopamine action 6-OHDA- induced neurodegeneration of neurons is also, in particular restoring mitochondrial function provides a useful model of Parkinson's disease (Wei et al (2015) Translational Neurodegeneration 4:. 11 ).

Thus, this study examines the effect of Quiuein on rat primary midbrain cultures damaged by exposure to 6-OHDA. Brain-derived neurotrophic factor (BDNF) has been proposed to reduce 6-OHDA-induced neurodegeneration in vitro and is used as a positive control.

1. Materials and Methods

1.1. compound

6-OHDA is obtained from Sigma (ref: H116).

Quieuins are described in Brooks et al . (2010) Tetrahedron Letters 51 : 4163-4165] and the thesis by Allen Brooks of the title " Synthesis of Tritium Labeled Queuine, PreQ1 and Related Azide Probes Toward Examining the Prevalence of Queuine" (2012, University of Michigan). .

BDNF is obtained from PanBiotech (Ref: CB-1115002).

In the following experiments 6-OHDA, Quieuin and BDNF are suspended in culture medium as defined below.

1.2. Rat Primary Culture of Dopamine Functional Neurons

Rat dopaminergic neurons were described in Schinelli et al . (1988) J. Neurochem . 50 : 1900-1907.

Briefly, pregnant female rats (Wistar rats; Janvier) at 15 days of gestation are killed by cervical dislocation and the fetus is removed from the uterus. The embryonic midbrain is removed and placed in ice-cold Leibovitz medium (L15; PanBiotech) containing 2% penicillin-streptomycin (PS; PanBiotech) and 1% bovine serum albumin (BSA; PanBiotech). Since it is a brain development site rich in dopamine-functional neurons, only the ventral portion of the midbrain flexion is used for cell production. The middle brain is separated by trypsinization (trypsin EDTA 1X; PanBiotech) at 37 ° C. for 20 minutes. The reaction was stopped by addition of Dulbecco's modified Eagle's medium (DMEM; PanBiotech) containing DNAse I Grade II (0.1 mg / ml; Roche Diagnostic) and 10% calf fetal serum (FCS; Invitrogen). Let's do it. Cells are mechanically separated by three passages through a 10 ml pipette. The isolated cells are then centrifuged at 180 × g for 10 minutes at 4 ° C. on a layer of BSA (3.5%) in L15 medium. The supernatant was discarded and pelleted cells were replaced with Neurobasal (Invitrogen, B27 (2%; Invitrogen, ref: 17504)), L-glutamine (2 mM; PanBiotech, Ref: P04-80100) and 2% PS. Resuspend in culture medium consisting of Ref: 21103, batch: 1725098), 10 ng / ml BDNF (PanBiotech, Ref: CB-1115002) and 1 ng / ml GDNF (PanBiotech, Ref: CB-1116001). The trypan blue exclusion test is used to count viable cells in the Neubauer cytometer. The cells are then seeded at a density of 40 000 cells / well in 96 well-plates precoated with poly-D-lysine (Greiner) and at 37 ° C. in a humid air (95%) / CO 2 (5%) atmosphere. Incubate. Half of the medium is replaced with fresh medium every two days. In these conditions, after 5 days of culture, astrocytes are present in the culture, releasing growth factors that allow neuronal differentiation. Five to six percent of neuronal cell populations are dopaminergic neurons.

1.3. Evaluation of Neuroprotective Effects on Rat Dopamine Functional Neurons

To evaluate the neuroprotective effect of Quiuein in 6-day culture according to the following protocol:

- Addition of control medium (culture medium);

- Addition of 6-OHDA (20 μΜ, 48H) + culture medium;

- Addition of 6-OHDA (20 μΜ, 48H) + BDNF (50 ng / ml);

- Addition of 6-OHDA (20 μΜ, 48H) + Quieuine (7 concentrations from 0.03 μΜ to 30 μΜ) added at three different times before, during or after 6-OHDA intoxication:

o 6 days before 6-OHDA poisoning, the compound will stay in the medium until poisoning;

o 1 day before 6-OHDA poisoning;

o 1 day after poisoning

The same conditions without 6-OHDA (un-addicted) are tested simultaneously to assess the effect of Quiuein on dopaminergic neuron survival.

Each condition is repeated in 6 wells of a 96-well culture plate.

1.4. Endpoint Evaluation: Determining the Total Number of Dopamine Functional Neurons

At the end of the incubation time, the cells are fixed by a solution of 4% paraformaldehyde for 20 minutes at room temperature. Cells are then permeabilized and blocking non-specific sites with a solution of phosphate buffered saline (PBS; PanBiotech) containing 0.1% saponin (Sigma) and 1% calf fetal serum (FCS) for 15 minutes at room temperature. do. Finally, cells are incubated with monoclonal anti-tyrosine hydroxylase (TH) mouse antibody (Sigma) in PBS containing 1% FCS and 0.1% saponin for 2 hours at room temperature. Anti-TH antibodies target dopaminergic neurons.

The antibody is revealed with Alexa Fluor 488-labeled goat anti-mouse IgG (Molecular probe) in PBS containing 1% FCS, 0.1% saponin for 1 hour at room temperature. The nuclei of cells are stained with fluorescent markers (Hoechst solution, SIGMA) in the same solution.

For each condition, 20 pictures are taken per well using InCell Analyzer ^™ 2000 (GE Healthcare) at 20 × magnification. Images of each culture well are also taken.

Cell sieve analysis of TH positive neurons is performed using Developer software (GE healthcare). A total of six data are provided per experimental condition. All values are expressed as mean ± mean standard error (s.e. mean). For statistical analysis, ANOVA is performed according to Dunnett's test.

2. Results

Preliminary results indicate that Quiuein has a neuroprotective effect on 6-OHDA-induced neurotoxicity.

As such, according to FIGS. 1 and 2 , 6-OHDA applied at 20 μM for 48 hours induces a large and significant reduction of TH positive neurons. Application of BDNF (50 ng / ml) 1 and 6 days before 6-OHDA poisoning showed a protective effect against 6-OHDA damage (*, p <0.05 in FIG. 1 , 80.80% compared to control, and FIG. * At 2 , p <0.05, 83.44% compared to control). These results confirm the study.

Moreover, FIG. 1 shows that when added before 1 day (24 hours) of 6-OHDA poisoning, the Quiuein at 1 μM shows a significant protective effect against 6-OHDA (*, p <0.05, 81.33% compared to the control). In addition, Figure 2 shows that, when added 6 days before 6-OHDA poisoning, Quieuin at 0.3 μM has a significant protective effect against 6-OHDA poisoning (*, p <0.05, compared to the control) 80.47%).

Claims (16)

A compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, for use in the prevention or treatment of a disease associated with mitochondrial dysfunction in an individual:

[In meals:
R ₁ represents -H or a ribosil group of the formula:

{In the expression:
R ₆ is -H; -OR ₉ or -O-CO-R ₉ , wherein R ₉ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms;
R ₇ is -H; -OR ₁₀ or -O-CO-R ₁₀ (R ₁₀ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms); Deoxyribonucleic acid groups; Or a ribonucleic acid group;
R ₈ is -H; -OR ₁₁ or -O-CO-R ₁₁ (R ₁₁ is H, an alkyl group having 1 to 20 carbon atoms or an aryl group having 3 to 20 carbon atoms); Phosphate groups; Diphosphate groups; Triphosphate groups; Deoxyribonucleic acid groups; Or a ribonucleic acid group};
R ₁₂ represents a saturated or unsaturated alkyl, cycloalkyl, heterocycloalkyl or ether group having 1 to 20 carbon atoms, optionally substituted by at least one group selected from the group consisting of:
Alkyl groups having 1 to 20 carbon atoms,
Aryl or heteroaryl groups having 3 to 20 carbon atoms,
A cycloalkyl or heterocycloalkyl group having 3 to 20 carbon atoms,
Hydroxyl group,
Carbonyl or carboxyl groups having from 1 to 20 carbon atoms,
ㆍ epoxy group,
A -OR ₄ group (R ₄ is H, an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group),
-O-CO-R ₅ group (R ₅ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms or a glycosyl group). The compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, according to claim 1, wherein the compound of formula (A) is of formula (I):

[In meals:
a represents a double bond or an epoxy group,
R ₁ represents -H or a ribosil group of the formula:

{In the expression:
R ₆ is -H; -OR ₉ or -O-CO-R ₉ , wherein R ₉ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms;
R ₇ is -H; -OR ₁₀ or -O-CO-R ₁₀ (R ₁₀ is H, an alkyl group having 1 to 6 carbon atoms or an aryl group having 3 to 12 carbon atoms); Deoxyribonucleic acid groups; Or a ribonucleic acid group;
R ₈ is -H; -OR ₁₁ or -O-CO-R ₁₁ (R ₁₁ is H, an alkyl group having 1 to 20 carbon atoms or an aryl group having 3 to 20 carbon atoms); Phosphate groups; Diphosphate groups; Triphosphate groups; Deoxyribonucleic acid groups; Or a ribonucleic acid group};
The same or different R ₂ and R ₃ are —OR ₄ (R ₄ is H, an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms, a glycosyl group or an aminoacyl group); Or —O—CO—R ₅ (R ₅ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 3 to 12 carbon atoms or a glycosyl group). The compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, according to claim 1 or 2, wherein:
The same or different R ₂ and R ₃ represent —OH, —O-mannosyl groups, —O-galactosyl groups or —O-glutamyl groups;
R ₆ represents -OH;
The same or different R ₇ and R ₈ represent —OH or ribonucleic acid groups. 4. The galactosyl-qui of claim 1, wherein the compound is quieuene, ent-quioue, keosine, epoxyquioue, epoxykeosine, mannosyl-quioue, galactosyl-quie A formula selected from the group consisting of euin, glutamyl-quiuein, galactosyl-keosin, mannosyl-keosin, glutamyl-keosin, quieuin-tRNA and epoxyquieuin-tRNA A compound of A), or a pharmaceutically acceptable salt or hydrate thereof. The compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, according to any one of claims 1 to 4, wherein the compound is selected from the group consisting of compounds of the formula:

. The compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, according to any one of claims 1 to 5, wherein the disease associated with mitochondrial dysfunction is a neurodegenerative disease associated with mitochondrial dysfunction. The compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, according to any one of claims 1 to 6, wherein the disease associated with mitochondrial dysfunction is Parkinson's disease. The compound of formula (A), or a pharmaceutically acceptable salt or hydrate thereof, according to any one of claims 1 to 7, for administration in a dosage regimen of from 0.01 to 40 mg / kg / d. The compound of formula (A) according to any one of claims 1 to 8, or a pharmaceutically acceptable form thereof, which is in a form suitable for administration by oral route, intradermal route, intravenous route, intramuscular route or subcutaneous route. Acceptable salts or hydrates. The compound of formula (A), or a pharmaceutically acceptable thereof according to any one of claims 1 to 9, in combination with at least one additional compound useful for the prevention or treatment of a disease associated with mitochondrial dysfunction. Salts or hydrates. Claim 1, together with at least one pharmaceutically acceptable excipient or vehicle, for use in the prophylaxis or treatment of a disease associated with mitochondrial dysfunction in claim 1, 6 or 7 A pharmaceutical composition comprising as an active substance a compound of formula (A) according to any one of claims 5 to 5, or a pharmaceutically acceptable salt or hydrate thereof. The method of claim 1, further comprising at least one additional compound useful for the prevention or treatment of a disease associated with mitochondrial dysfunction, optionally in combination with at least one pharmaceutically acceptable excipient or vehicle. A pharmaceutical composition comprising a compound of formula (A) according to claim 1, or a pharmaceutically acceptable salt or hydrate thereof as an active substance. The pharmaceutical preparation of claim 12 for use in the prevention or treatment of a disease associated with mitochondrial dysfunction in claim 1, 6 or 7 in an individual. Relic. As a combined agent for simultaneous, separate or sequential use in the prevention or treatment of a disease associated with mitochondrial dysfunction in claim 1, 6 or 7, in an individual:
-A compound of formula (A) according to any one of claims 1 to 5,
At least one additional compound useful for the prevention or treatment of diseases associated with mitochondrial dysfunction
Product comprising. A compound of formula (A) according to any one of claims 1 to 5 for use in reducing the risk of a disease associated with mitochondrial dysfunction in an individual. Health supplements containing. 16. The dietary supplement of claim 15 comprising an additional compound selected from the group consisting of vitamins, minerals, fatty acids, amino acids and antioxidants.

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